In our current IPCAVD program (U19 AI096040), we have developed alternative serotype adenovirus vectors, bioinformatically optimized HIV-1 mosaic antigens, and stable Env gp140 protein immunogens. We have shown that the Ad26-Env/Gag/Pol prime, Env gp140 protein boost (Ad26/Env) vaccine afforded unprecedented protective efficacy against acquisition of infection following heterologous SIVmac251 challenges in rhesus monkeys, as well as robust protection against SHIV-SF162P3 challenges. Protection correlated with polyfunctional antibody responses as determined by systems serology. We have also advanced the Ad26/Env, Ad26/MVA, and Ad26/MVA/Env mosaic vaccines into phase 1/2a clinical trials in the United States, East Africa, South Africa, and Thailand. Over the next 5 years of this IPCAVD program, we propose to define the extent and mechanism of protection achieved by our lead Ad26/Env vaccine regimen in rhesus monkeys, to advance our lead vaccine regimen into phase 2b/3 efficacy studies in humans, and to develop an improved next generation HIV-1 vaccine candidate. In this Project, we hypothesize that our current Ad26/Env mosaic vaccine as well as simplified vaccine regimens will protect against SHIVs derived from diverse clades by a consistent mechanism involving polyfunctional non-neutralizing antibodies. We further hypothesize that a next generation HIV-1 vaccine candidate that elicits augmented NAb magnitude and breadth will improve protective efficacy against SHIV challenges in rhesus monkeys. To test these hypotheses we propose the following Specific Aims: Specific Aim 1. To define the protective efficacy and protective mechanism of our Ad26/Env mosaic vaccine and simplified vaccine regimens against SHIVs derived from diverse clades in rhesus monkeys Specific Aim 2. To develop an improved HIV-1 vaccine strategy that elicits augmented NAb responses in rhesus monkeys